The molecular mechanisms by which synapses form during development and are modified in the adult are largely unknown. Understanding the mechanisms by which cell-cell interactions at the synapse influence synaptic protein expression will provide insight into synaptogenesis and synapse modulation. The long term goal of this research application is to characterize these mechanisms. The neuromuscular junction provides an ideal system for studying these processes since it is homogenous, accessible to experimental manipulation and is the best studied synapse in the nervous system. The nicotinic acetylcholine receptor (nAChR) mediates communication across this synapse and serves as an excellent marker for studying pre- and post-synaptic cell interactions regulating synaptic protein expression. During development, nerve-induced muscle electrical activity suppresses nAChR gene expression in extrajunctional regions of the muscle fiber, while nerve-derived factors induce nAChR gene expression at the endplate. In this application we propose to focus on activity-dependent control of nAChR gene expression. Protein kinase C (PKC) is proposed to mediate activity-dependent suppression of nAChR gene expression in birds, however no such evidence exists for mammalian muscle. We will characterize PKC activity in active and inactive muscle and determine if inhibition or activation of specific PKC isoforms can influence nAChR gene expression in mammalian muscle. Calcium suppresses nAChR gene expression via DNA sequences mediating activity-dependent regulation. Calcium's effect appears to be independent of PKC and may be mediated, in part, by calcium/calmodulin-dependent protein kinase. Therefore we will characterize the mechanism by which calcium regulates nAChR gene expression and determine if this regulation also participates in activity-dependent control of these genes. In addition to myogenin, SP1 also activates nAChR gene expression and may interact with myogenin. We propose to characterize the mechanism by which SP1 mediates increased nAChR gene expression and if this SP1-dependent regulation is controlled by muscle activity. Finally, we will determine if myogenin and SP1 phosphorylation is regulated by increased intracellular calcium and muscle depolarization and we will map these putative phosphorylation sites.